Porphyrin binding mechanism is altered by protonation at the loops in G-quadruplex DNA formed near the transcriptional activation site of the human c-kit gene

Background G-quadruplex DNA structures are hypothesized to be involved in the regulation of gene expression and telomere homeostasis. The development of small molecules that modulate the stability of G-quadruplex structures has a potential therapeutic interest in cancer treatment and prevention of aging. Methods Molecular absorption and circular dichroism spectra were used to monitor thermal denaturation, acid base titration and mole ratio experiments. The resulting data were analyzed by multivariate data analysis methods. Surface plasmon resonance was also used to probe the kinetics and affinity of the DNA-drug interactions. Results We investigated the interaction between a G-quadruplex-forming sequence in the human c-kit proto-oncogene and the water soluble porphyrin TMPyP4. The role of cytosine and adenine residues at the loops of G-quadruplex was studied by substitution of these residues by thymidines. Conclusions Here, we show the existence of two binding modes between TMPyP4 and the considered G-quadruplex. The stronger binding mode (formation constant around 107) involves end-stacking, while the weaker binding mode (formation constant around 106) is probably due to external loop binding. Evidence for the release of TMPyP4 upon protonation of bases at the loops has been observed. General significance The results may be used for the design of porphyrin-based anti-cancer molecules with a higher affinity to G-quadruplex structures which may have anticancer properties. Graphical abstract Protonation pushes away TMPyP4 molecules from the loops in G-quadruplex structures. The interaction of TMPyP4 porphyrin with the G-quadruplex structure formed by a guanine-rich sequence in the promoter region of c-kit gene was studied. Up to three ligand molecules may be bound to the G-quadruplex structure. Protonation at the loops induces the release of one TMPyP4 molecule

Vibrational spectroscopic image analysis of biological material using multivariate curve resolution - alternating least squares

Multivariate data analysis techniques are ideal to decrypt chemical differences between anatomical features or tissue areas in hyperspectral images of biological samples. This protocol provides a user-friendly pipeline and graphical user interface (GUI) for data pre-processing and un-mixing of pixel spectra into their contributing pure components by multivariate curve resolution-alternating least squares (MCR-ALS) analysis. The analysis considers the full spectral profile to identify the chemical compounds and to visualize their distribution across the sample to categorize chemically distinct areas. Results are rapidly achieved (usually less than 30 - 60 min/image) and are easy to interpret and evaluate both in terms of chemistry and biology, making the method generally more powerful than principal component analysis (PCA) or single band intensity heap maps. In addition, chemical and biological evaluation of the results by means of reference matching and segmentation maps (based on k-means clustering) are possible

Resolució multivariant en Química: a la cerca de la bella simplicitat de la mesura

En Quimiometria, existeix una família de mètodes, coneguts sota la denominació comuna de resolució multivariant, que permeten descriure fenòmens molt complexos i diversos a partir de la combinació de poques contribucions bàsiques fàcilment interpretables. La versatilitat i simplicitat dels models de resolució multivariant ha fet que s'hagin aplicat amb èxit en el modelatge de processos d'origen químic i bioquímic i en la interpretació de dades -òmiques i ambientals

The human mitochondrial transcription factor A is a versatile G-quadruplex binding protein

The ability of the guanine-rich strand of the human mitochondrial DNA (mtDNA) to form G-quadruplex structures (G4s) has been recently highlighted, suggesting potential functions in mtDNA replication initiation and mtDNA stability. G4 structures in mtDNA raise the question of their recognition by factors associated with the mitochondrial nucleoid. The mitochondrial transcription factor A (TFAM), a highmobility group (HMG)-box protein, is the major binding protein of human mtDNA and plays a critical role in its expression and maintenance. HMG-box proteins are pleiotropic sensors of DNA structural alterations. Thus, we investigated and uncovered a surprising ability of TFAM to bind to DNA or RNA G4 with great versatility, showing an affinity similar than to double-stranded DNA. The recognition of G4s by endogenous TFAM was detected in mitochondrial extracts by pull-down experiments using a G4-DNA from the mtDNA conserved sequence block II (CSBII). Biochemical characterization shows that TFAM binding to G4 depends on both the G-quartets core and flanking single-stranded overhangs. Additionally, it shows a structure-specific binding mode that differs from B-DNA, including G4- dependent TFAM multimerization. These TFAM-G4 interactions suggest functional recognition of G4s in the mitochondria

Metabolic profiling for the identification of Huntington biomarkers by on-line solid-phase extraction capillary electrophoresis mass spectrometry combined with advanced data analysis tools

In this work, an untargeted metabolomic approach based on sensitive analysis by on-line solid-phase extraction capillary electrophoresis mass spectrometry (SPE-CE-MS) in combination with multivariate data analysis is proposed as an efficient method for the identification of biomarkers of Huntington's disease (HD) progression in plasma. For this purpose, plasma samples from wild type (wt) and HD (R6/1) mice of different ages (8, 12 and 30 weeks), were analysed by C18-SPE-CE-MS in order to obtain the characteristic electrophoretic profiles of low molecular mass compounds. Then, multivariate curve resolution alternating least squares (MCR-ALS) was applied to the multiple full scan MS data sets. This strategy permitted the resolution of a large number of metabolites being characterised by their electrophoretic peaks and their corresponding mass spectra. A total number of 29 compounds were relevant to discriminate between wt and HD plasma samples, as well as to follow-up the HD progression. The intracellular signalling was found to be the most affected metabolic pathway in HD mice after 12 weeks of birth, when mice already showed motor coordination deficiencies and cognitive decline. This fact agreed with the atrophy and dysfunction of specific neurons, loss of several types of receptors and changed expression of neurotransmitters

Nous desenvolupaments, aplicacions bioanalítiques i validació dels mètodes de resolució multivariant

[cat] Aquest treball s'integra en una de les línies d'investigació del grup de recerca "Quimiometria" del Departament de Química Analítica de la Universitat de Barcelona. Aquesta línia d'investigació es centra en el desenvolupament de mètodes quimiomètrics d'anàlisi multivariant de dades, i en la seva aplicació a l'estudi analític dels canvis de conformació i/o de les interaccions entre biomolècules. Actualment és possible enregistrar l'espectre sencer d'una mostra en poc temps. Aquest augment del nombre i de la complexitat de les dades adquirides ha portat a l'aparició de mètodes que tenen com a finalitat la obtenció d'informació d'interés físico-químic a partir d'aquests conjunt de dades. Amb aquesta finalitat es poden trobar dues aproximacions: a) els mètodes de modelatge rígid que exigeixen la postulació d'un model químic o cinètic al qual ajustar les dades experimentals, i b) els mètodes de modelatge flexible que no necessiten la postulació d'un model. El treball realitzat en aquests tesi doctoral es pot dividir en tres blocs. En primer lloc, s'ha desenvolupat una interfície gràfica en l'entorn de programació MATLAB pel mètode de resolució multivariant de corbes mitjançant mínims quadrats alternats (MCR-ALS). Aquesta interfície millora notablement la interacció entre l'usuari i el programa, i potencía la seva utilització generalitzada per part d'usuaris no acostumats a treballar amb eines pròpies de la Quimiometria. En segon lloc, s'ha dut a terme la validació de diversos mètodes d'anàlisi multivariant, és a dir, s'ha estudiat la fiabilitat de les solucions obtingudes per aquest tipus de mètodes quimiomètrics. Així, pel mètode MCR-ALS, s'ha analitzat la influència i la propagació de l'error experimental i les possibles repercusions sobre les ambigüetats matemàtiques existents en les solucions obtingudes. Aquest estudi s'ha realitzat tant en el cas de l'anàlisi individual de matrius de dades obtingudes en un únic experiment, com en el cas de l'anàlisi simultani de matrius de dades obtingudes en diversos experiments. En el cas dels mètodes de modelatge rígid s'ha estudiat l'ambigüetat existent al ajustar mecanismes cinètics complexos. En aquest cas s'ha observat l'aparició de mínims locals múltiples amb el mateix valor d'ajust en la superfície de desposta associada. Finalment, s'han aplicat els mètodes quimiomètrics de modelatge flexible i de modelatge rígid a l'estudi dels equilibris en solució dels àcids nucleics. Aquestes són biomolècules que tenen una organització jeràrquica començant en la seqüència de nucleòtids a les cadenes fins a estructures complexes d'ordre superior com els tríplexs o quadruplexs. Els canvis conformacionals o les interaccions amb d'altres biomolècules s'han estudiat tradicionalment mitjançant experiments seguits amb tècniques espectroscòpies. En aquest treball es seguiran aquests processos mitjançant lectures a moltes longituts d'ona (aproximació multivariant) i s'aplicaran mètodes quimiomètrics adients de tractaments de dades multivariants. Els procesos estudiants en aquesta Tesi són bàsicament els canvis conformacionals provocats en variar condicions del medi, com el pH, la temperatura, la concentració d'altres ions... S'han emprat tècniques espectroscòpiques com l'absorció molecular a l'UV-visible, la fluorescència, el dicroisme circular i la ressonància magnètica nuclear. Una altra aplicació, ha estat l'anàlisi de micromatrius d'ADN. L'aparició d'aquesta la tecnologia ha permès obtenir informació sobre els nivells de l'expressió gènica per un gran nombre de gens en un únic experiment. La generació de grans quantitats de dades requereix la utilització d'eines mitjançant les quals es pugui extreure la informació biològica. En aquest treball s'ha aplicat el mètode MCR-ALS a l'anàlisis de diversos conjunts de dades per tal de poder determinar la relació entre les mostres que presenten diferents tipus de càncer i els gens estudiats.[eng] This PhD Thesis has been developed in the framework of the Chemometrics group at the Universitat de Barcelona. The work deals with the development and validation of Multivariate Curve Resolution (MCR) methods (both hard- and soft-modelling), and with their application to bioanalytical problems. The work has been organized into three blocks: First, a graphical interface has been developed for the program running the MCR-ALS (Multivariate Curve resolution Alternating Least Squares) method in the MATLAB® environment. This interface improves the interaction between the user and the program and facilitates the use of multivariate curve resolution to little experineced potential users. Secondly, validation of multivariate resolution methods of data analysis has been carried out. For the MCR-ALS method, effects of rotational ambiguities and of propagation of experimental noise have been studied. These studies have been performed in the analysis of a single experiment and in the case of analyzing multiple experiments simultaneously. In the case of hard-modelling kinetic data fitting methods, ambiguities in the analysis of kinetic experiments have been studied and methods to overcome this ambiguity have been proposed. Third, multivariate resolution methods have been applied to the study of conformational equilibria of nucleic acids. These are biomolecules that have a hierarchic organization from the nucleotide sequence to higher order structures such as triplex or quadruplex. Traditionally, conformational changes or interactions of nucleic acids with other biomolecules have been spectroscopically monitored at just one wavelength. In this work, these processes have been followed at multiple wavelengths and suitable multivariate resolution methods for the data treatment have been applied. Processes studied during this Thesis have been DNA conformational changes induced by pH, temperature or salinity. Spectroscopic techniques such as molecular absorption in the UV-visible, circular dichroism or nuclear magnetic resonance have been used for this purpose. Finally, data obtained using DNA microarrays have been analyzed. This technique allows highthroughput analysis of relative gene expressions of thousands of genes of an organism that generates large amounts of data. This has caused a need for statistical methods that can extract useful information for further research. In this PhD Thesis, the MCR-ALS method has been proposed for the analysis of this kind of data with very promising results

Multivariate curve resolution: a powerful tool for the analysis of conformational transitions in nucleic acids

A successful application is reported of the multivariate curve resolution alternating least‐squares method (MCR‐ALS) for the analysis of nucleic acid melting and salt‐induced transitions. Under conditions where several structures co‐exist in a conformational equilibrium, MCR‐ALS analysis of the UV and circular dichroism (CD) spectra at different temperatures, ionic strength and oligonucleotide concentration allows for the resolution of concentration profiles and pure spectra of the different species. The methodology is illustrated by the case of the cyclic oligonucleotide d. The melting transition of this molecule at different oligonucleotide concentrations was studied at 0, 2 and 10 mM MgCl 2 by UV and CD spectroscopy. In addition, salt titration experiments were carried out at 21.0 and 54.0°C. The MCR‐ALS analysis indicates that three different conformations of this molecule co‐exist in solution. In agreement with previous NMR studies, these conformations were assigned to a monomeric dumbbell‐like structure, a dimeric four‐stranded conformation and a disordered (random coil) structure. The MCR‐ALS methodology allows for a detailed analysis of how this equilibrium is affected by temperature, salt and oligonucleotide concentration

Identification of antihypertensive peptides in nutraceuticals by capillary electrophoresis-mass spectrometry

We present capillary electrophoresis-mass spectrometry (CE-MS) in combination with advanced chemometric tools for the analysis of bioactive compounds in food, in particular for the identification of antihypertensive peptides in a nutraceutical derived from a bovine milk protein hydrolysate. Different extracts of the nutraceutical were analyzed by CE-MS, and the electropherograms were processed using a novel data analysis workflow that included regions of interest (ROIs) compression and multivariate curve resolution alternating least squares (MCR-ALS). MCR-ALS permitted the description of the nutraceutical extract as ten characteristic components with their electrophoretic profiles and mass spectra. Twenty-two compounds were tentatively identified as antihypertensive bovine casein fragments through a mass search in a database of bioactive peptides. The identity of 17 antihypertensive peptides was reliably confirmed by capillary electrophoresis-tandem mass spectrometry. The proposed analytical approach demonstrated the potential to obtain a characteristic and activity-related fingerprint for quality control and authentication of the antihypertensive nutraceutical

Porphyrin binding mechanism is altered by protonation at the loops in G-quadruplex DNA formed near the transcriptional activation site of the human c-kit gene

Background G-quadruplex DNA structures are hypothesized to be involved in the regulation of gene expression and telomere homeostasis. The development of small molecules that modulate the stability of G-quadruplex structures has a potential therapeutic interest in cancer treatment and prevention of aging. Methods Molecular absorption and circular dichroism spectra were used to monitor thermal denaturation, acid base titration and mole ratio experiments. The resulting data were analyzed by multivariate data analysis methods. Surface plasmon resonance was also used to probe the kinetics and affinity of the DNA-drug interactions. Results We investigated the interaction between a G-quadruplex-forming sequence in the human c-kit proto-oncogene and the water soluble porphyrin TMPyP4. The role of cytosine and adenine residues at the loops of G-quadruplex was studied by substitution of these residues by thymidines. Conclusions Here, we show the existence of two binding modes between TMPyP4 and the considered G-quadruplex. The stronger binding mode (formation constant around 107) involves end-stacking, while the weaker binding mode (formation constant around 106) is probably due to external loop binding. Evidence for the release of TMPyP4 upon protonation of bases at the loops has been observed. General significance The results may be used for the design of porphyrin-based anti-cancer molecules with a higher affinity to G-quadruplex structures which may have anticancer properties. Graphical abstract Protonation pushes away TMPyP4 molecules from the loops in G-quadruplex structures. The interaction of TMPyP4 porphyrin with the G-quadruplex structure formed by a guanine-rich sequence in the promoter region of c-kit gene was studied. Up to three ligand molecules may be bound to the G-quadruplex structure. Protonation at the loops induces the release of one TMPyP4 molecule

Exemple de volumetria directa

Aquest material forma part d'una col·lecció de tres vídeos, que estan creats per tal de mostrar a l'alumne de Química Analítica la diferència que hi ha entre una volumetria directa, una volumetria indirecta i una volumetria per retrocés. En aquest vídeo es mostra la volumetria directa.Universitat de Barcelona, Projecte d'Innovació Docent: 2012PID-UB/18